Heat exchanging, optically interrogated chemical reaction assembly

ABSTRACT

The present invention provides a reaction vessel and apparatus for performing heat-exchanging reactions. The vessel has a chamber for holding a sample, the chamber being defined by a plurality of walls, at least two of the walls being light transmissive to provide optical windows to the chamber. The apparatus comprises at least one heating surface for contacting at least one of the plurality of walls, a heat source for heating the surface, and optics positioned to optically interrogate the chamber while the heating surface is in contact with at least one of the plurality of walls. The optics include at least one light source for transmitting light to the chamber through a first one of the light transmissive walls and at least one detector for detecting light exiting the chamber through a second one of the light transmissive walls.

RELATED APPLICATION INFORMATION

This application is a national stage entry (371) of InternationalApplication PCT/US98/03962 filed Mar. 2, 1998 which internationalapplication is a continuation from U.S. Ser. No. 08/808,325 filed Feb.28, 1997, now U.S. Pat. No. 5,958,349, U.S. Ser. No. 08/808,327 filedFeb. 28, 1997, now abandoned, and U.S. Ser. No. 08/808,733 filed Feb.28, 1997, now abandoned.

This invention was made with Government support under contractDAAM01-96-C-0061 awarded by the U.S. Army. The Government has certainrights in the invention.

FIELD OF THE INVENTION

The present invention provides an assembly useful for heat exchangingchemical processes.

BACKGROUND OF THE INVENTION

There are many applications in the field of chemical processing in whichit is desirable to precisely control the temperature of chemicals and toinduce rapid temperature transitions. In these reactions, heat isexchanged between chemicals and their environment to increase ordecrease the temperature of the reacting chemicals. It is oftendesirable to control the temperature change in a manner that accuratelyattains the target temperature, avoids undershooting or overshooting ofthe temperature, and quickly reaches the target temperature. Suchcontrol of temperature may inhibit side reactions, the formation ofunwanted bubbles, the degradation of components at certain temperatures,etc., which may occur at non-optimal temperatures. It is of furtherinterest to optically observe and monitor the chemical reaction.

Applications for heat exchanging chemical reactions may encompassorganic, inorganic, biochemical and molecular reactions, and the like.In organic and inorganic reactions, chemicals may be heated to achievethe activation energy for the reaction. Examples of thermal chemicalreactions include isothermal nucleic acid amplification, thermal cyclingamplification, such as polymerase chain reaction (PCR), ligase chainreaction (LCR), self-sustained sequence replication, enzyme kineticstudies, homogenous ligand binding assays, and more complex biochemicalmechanistic studies that require complex temperature changes.Temperature control systems also enable the study of certain physiologicprocesses where a constant and accurate temperature is required.

Numerous devices and systems have been described in the art forconducting thermal transfer reactions. These devices use a variety ofdesigns for heat transfer, such as water baths, air baths, and solidblocks such as aluminum. Chemical reactions in small reaction volumeshave also been described.

Conventional instrumentation, for example, typically consists of a blockof aluminum having as many as ninety-six conical reaction tubes. Thealuminum block is heated and cooled either by a Peltier heating/coolingapparatus, or by a closed-loop liquid heating/cooling system, flowingthrough channels machined into the aluminum block. Because of the largethermal mass of the aluminum block, heating and cooling rates arelimited to about 1° C./sec resulting in longer processing times. Forexample, in the PCR application, fifty cycles may require two or morehours to complete.

Part of the reason for the relatively large metal block is to providesufficient mass to ensure a constant and uniform temperature at eachreaction site, as well as from site to site. Some chemical reactioninstruments also incorporate a top-plate, which is heated and cooled toensure a uniform temperature across the top of all sample solutions. Thesample inserts are tapered to maximize thermal contact between theinsert and the metal block. One problem with these instruments is thatthe large thermal masses, required for temperature uniformity, take along time (and or a large heating/cooling power source) to heat and tocool. Usual heating and cooling rates for these types of instruments areon the order of 1-3° C./second.

Typically, the highest heating rate obtainable in commercial instrumentsis on the order of 3° C./second, and cooling rates are significantlyless. With these relatively slow heating and cooling rates, it has beenobserved that some processes requiring high control of temperature areinefficient. For example, reactions may occur at the intermediatetemperatures, creating unwanted and interfering side products, such asin PCR “primer-dimers” or anomalous amplicons, which are deleterious tothe analytical process. The poor control of temperature also results inover consumption of reagents necessary for the intended reaction.

Furthermore, for some diagnostic and environmental chemical detectionmethodologies, the volume of the tested unknown sample can be important.For example, in the detection of viruses in blood or other bodily fluidsusing PCR, the detection limit is about 10 virions. Therefore, a minimumfluid volume is required depending upon the concentration of virions inthe sample. By way of illustration, at a concentration of 100virions/mL, the sample size should be at least 0.1 mL. For more dilutesamples, even larger sample volumes are necessary. Therefore, thechemical analysis system should be capable of handling milliliter fluidvolumes.

Another requirement in many chemical analyses is the ability to monitorthe chemical reaction and detect the resulting product. A preferreddetection technique is optical interrogation, typically usingfluorescence or chemiluminescence. For ligand-binding assays,time-resolved fluorescence and fluorescence polarization are often used.

The control of heating and cooling changes may be referred to as thermalcycling. The term “thermal cycling” is herein intended to mean at leastone change of temperature, i.e. increase or decrease of temperature, inthe environment to which chemicals are exposed. Therefore, chemicalsundergoing thermal cycling may shift from one temperature to another andthen stabilize at that temperature, transition to a second temperatureor return to the starting temperature. The temperature cycle may beperformed once or repeated as many times as required by the particularchemical reaction. The various chemical reactions occurring during thesetemperature cycles are more specific and more efficient when thetemperature is raised and lowered to the various required reactiontemperatures as quickly as possible and controlled very precisely.

Devices which control the transfer of heat for chemical reactions areapplicable for synthesis reactions such as thermal cycling PCR toamplify a segment of nucleic acid. In this methodology, a DNA templateis used with a thermostable DNA polymerase, e.g., Taq DNA polymerase,nucleoside triphosphates, and two oligonucleotides with differentsequences, complementary to sequences that lie on opposite strands ofthe template DNA and which flank the segment of DNA that is to beamplified (“primers”). The reaction components are cycled between ahigher temperature (e.g., 95° C.) for dehybridizing double strandedtemplate DNA, followed by lower temperatures (e.g., 40-60° C. forannealing of primers and 70-75° C. for polymerization). Repeated cyclingamong dehybridization, annealing, and polymerization temperaturesprovides exponential amplification of the template DNA. For example, upto 1 μg of target DNA up to 2 kb in length can be obtained with 30-35cycles of amplification from only 10⁻⁶ μg of starting DNA.

Amplification has been applied to the diagnosis of genetic disorders;the detection of nucleic acid sequences of pathogenic organisms in avariety of samples including blood, tissue, environmental, air borne,and the like; the genetic identification of a variety of samplesincluding forensic, agricultural, veterinarian, and the like; theanalysis of mutations in activated oncogenes, detection of contaminantsin samples such as food; and in many other aspects of molecular biology.Polynucleotide amplification assays can be used in a wide range ofapplications such as the generation of specific sequences of cloneddouble-stranded DNA for use as probes, the generation of probes specificfor uncloned genes by selective amplification of particular segments ofcDNA, the generation of libraries of cDNA from small amounts of mRNA,the generation of large amounts of DNA for sequencing and the analysisof mutations. Instruments for performing automated PCR chain reactionsvia thermal cycling are commercially available.

Some of the instrumentation suitable for newer processes, requiring“real-time” optical analysis after each thermal cycle, has only recentlybecome available. For example, the Perkin Elmer (PE) 7700 (ATC)instrument as well as the PE 9600 thermal cycler are based on a 96-wellaluminum block format, as described above. Optical fluorescencedetection in the PE 7700 is accomplished by guiding an optical fiber toeach of the ninety-six reaction sites. A central high power lasersequentially excites each reaction tube and captures the fluorescencesignal through the optical fiber. Complex beam-guiding and opticalmultiplexing are typically required.

A different thermal cycling instrument is available from IdahoTechnologies. This instrument employs forced-air heating and cooling ofcapillary sample carriers mounted in a carousel. The instrument monitorseach capillary sample carrier in sequence as the capillary samplecarriers are rotated past an optical detection site.

A third real-time PCR analysis system is the MATCI device developed byDr. Allen Northrup et al., as disclosed in U.S. Pat. No. 5,589,136,incorporated herein by reference. This device uses a modular approach toPCR thermal cycling and optical analysis. Each reaction.is performed inits own silicon sleeve and each sleeve has its own associated opticalexcitation source and fluorescence detector. The low thermal mass of thethermal cycling sleeve allows the MATCI device to realize fast thermalheating and cooling rates, up to 30° C./sec heating and 5° C./seccooling.

There are, however, disadvantages to this MATCI device in its use of amicromachined silicon sleeve that incorporates a heating elementdirectly deposited on the sleeve. A first disadvantage is that thebrittle silicon sleeve may crack and chip. A second disadvantage is thatit is difficult to micromachine a silicon sleeve and heating elementwith sufficient precision to allow the sleeve to precisely accept aplastic insert that holds the sample.

For the reasons stated above, optimization of many biochemical reactionprocesses, including the PCR process, require that the desired reactiontemperatures be reached as quickly as possible, spending minimal time atintermediate temperatures. Therefore, the heating and cooling system inwhich the sample reacts should permit rapid heating and cooling rates.It is also desirable that such a system permit real time opticalinterrogation of the sample.

SUMMARY

A reaction vessel and apparatus for performing heat-exchanged chemicalreactions are provided. The reaction vessel and apparatus are designedfor optimal thermal transfer to or from a sample and for efficientoptical viewing of a chemical reaction with the sample.

In accordance with an aspect of the present invention, the vessel has achamber for holding a reaction mixture, the chamber being defined by twoopposing major walls and a plurality of minor walls joining the majorwalls to each other. At least two of the minor walls are lighttransmissive to provide optical windows to the chamber. The apparatusincludes at least one heating surface for contacting at least one of themajor walls. The apparatus also includes optics for opticallyinterrogating the chamber while the heating surface is in contact withat least one of the major walls. The optics comprise at least one lightsource for transmitting light to the chamber through a first one of thelight transmissive walls and at least one detector for detecting lightexiting the chamber through a second one of the light transmissivewalls.

In some embodiments, the apparatus includes at least two heatingsurfaces defined by opposing plates positioned to receive the vesselbetween them such that the plates contact the major walls, heatingresistors are coupled to the plates, and the optics are positioned tointerrogate the chamber through at least one window or opening betweenthe plates. In some embodiments, the optics include a plurality of lightsources and filters for transmitting different wavelengths of excitationlight to the chamber; and a plurality of detectors and filters fordetecting different wavelengths of light emitted from the chamber.

In accordance with another aspect of the present invention, an apparatusfor controlling the temperature of a sample comprises a vessel having achamber defined by two opposing major walls and a plurality of rigidminor walls joining the major walls to each other. At least one of themajor walls comprises a sheet or film. The vessel also includes a portfor introducing fluid into the chamber, and a channel connecting theport to the chamber. The apparatus also includes at least one heatingsurface for contacting the sheet or film, the sheet or film beingsufficiently flexible to conform to the surface. The apparatus furtherincludes a plug that is insertable into the channel to increase pressurein the chamber, whereby the pressure increase in the chamber forces thesheet or film against the heating surface.

In accordance with another aspect of the present invention, there isprovided an apparatus for heating and optically interrogating a reactionmixture contained in a vessel. The vessel has a chamber for holding themixture, the chamber being defined by a plurality of walls, and at leasttwo of the walls are light transmissive to provide optical windows tothe chamber. The apparatus comprises at least one heating surface forcontacting at least one of the plurality of walls; at least one heatsource for heating the surface; and optics positioned to opticallyinterrogate the chamber while the heating surface is in contact with atleast one of the plurality of walls. The optics comprise at least onelight source for transmitting light to the chamber through a first oneof the light transmissive walls and at least one detector for detectinglight exiting the chamber through a second one of the light transmissivewalls.

In accordance with another aspect of the present invention, there isprovided an apparatus for heating and optically interrogating a reactionmixture contained in a vessel, the vessel having a chamber defined bytwo opposing major walls and a plurality of minor walls joining themajor walls to each other. At least two of the walls defining thechamber are light transmissive to provide optical windows to thechamber. The apparatus comprises at least one plate for contacting atleast one of the major walls; at least one heater for heating the plate;and optics positioned to optically interrogate the contents of thechamber while the plate is in contact with at least one of the majorwalls. The optics comprise at least one light source for transmittinglight to the chamber through a first one of the light transmissive wallsand at least one detector for detecting light exiting the chamberthrough a second one of the light transmissive walls.

Another aspect of the present invention is directed to a vessel having areaction chamber for holding a sample. The vessel comprises a rigidframe defining the minor walls of the chamber. At least two of the minorwalls are light transmissive to provide optical windows to the chamber.At least one sheet or film is attached to the rigid frame to form amajor wall of the chamber. The vessel also includes a port forintroducing the sample into the chamber.

In some embodiments, the vessel includes at least two sheets or filmsthat are attached to opposite sides of the frame to form two opposingmajor walls of the chamber, each of the sheets or films beingsufficiently flexible to conform to a respective heating surface. Insome embodiments, the light transmissive walls are angularly offset fromeach other, preferably by about 90°.

Another aspect of the present invention is directed to a vessel having areaction chamber for holding a sample. The vessel comprises a rigidframe defining the minor walls of the chamber. At least one sheet orfilm is attached to the rigid frame to form a major wall of the chamber.The vessel also comprises a port for introducing the sample into thevessel; a channel connecting the port to the chamber; and a plug that isinsertable into the channel to increase pressure in the chamber in someembodiments, the vessel includes at least two sheets or films that areattached to opposite sides of the frame to form two opposing major wallsof the chamber, each of the sheets or films being sufficiently flexibleto conform to a respective heating surface.

Another aspect of the present invention is directed to a vesselcomprising two opposing major walls and a plurality of rigid minor wallsjoining the major walls to each other to form a reaction chamber. Atleast one of the major walls comprises a sheet or film, and at least twoof the minor walls are light transmissive. The vessel includes a portfor introducing fluid into the chamber. In some embodiments, the ratioof the total surface area of the major walls to that of the minor wallsis at least 2:1. In some embodiments, the ratio of the thermalconductance of the major walls to that of the minor walls is at least2:1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a partially exploded, isometric view of a reaction vesselhaving a reaction chamber, wherein two walls of the reaction chamber areremoved to show the interior of the chamber.

FIGS. 2a, b show schematic, side views of a heat-exchanging module witha reaction vessel and thermal sleeve. FIG. 2a shows the module prior tobiasing the sleeve against the vessel, and FIG. 2b shows the moduleafter the sleeve is made to bias against the inserted vessel.

FIG. 3 shows a plan view of a reaction vessel inserted in a thermalsleeve having a heating element and cooling unit according to oneembodiment of the present invention.

FIGS. 4a, b, c, d show various views of another embodiment of a reactionvessel according to the present invention. FIG. 4a shows a side view ofthe vessel, FIG. 4b shows a front view of the vessel, FIG. 4c shows across sectional view of the vessel with a channel leading to a reactionchamber, and FIG. 4d shows a top view of the vessel.

FIG. 5 shows a schematic, side view of a reaction vessel with lenses foroptical detection in arrangement with light sources and opticaldetectors.

FIG. 6 shows a partially exploded, isometric view of a thermal sleevewith one heating plate attached to a support and, for illustrationpurposes, the other plate removed from the support.

FIG. 7 shows an isometric view of another heat exchanging assembly witha reaction vessel inserted in a thermal sleeve.

FIGS. 8a, b, c, d show various heating and cooling configurations of athermal sleeve according to alternative embodiments of the presentinvention. FIG. 8a is a top view of the heating and cooling elements ofa sleeve, FIG. 8b is a front view of the cooling elements shown in FIG.8a, FIG. 8c is a front view of another sleeve with heating and coolingelements, and FIG. 8d is a side view of the sleeve shown in FIG. 8c.

FIG. 9 shows an isometric view of a heat exchanging instrument having athermal sleeve, optics assembly coupled to circuit boards, and a coolingunit. A reaction vessel is exploded from the instrument.

FIGS. 10a, b show schematic views of a heat exchanging unit according toanother embodiment of the invention. FIG. 10a shows a front view of theunit, and FIG. 10b shows a side view of the unit.

FIGS. 11a, b and c show schematic views of a cluster of modular, heatexchanging units on a base support. FIG. 11a shows four unitsinterfacing with a main controller board of the base support, FIG. 11bshows one of the units of FIG. 11a in pneumatic and electricalcommunication with the base support, and FIG. 11c shows eight unitsinterfacing individual controller boards.

DETAILED DESCRIPTION

In general, this invention provides a low thermal mass heating andcooling assembly for the rapid heating and cooling of chemical solutionsto perform reactions and for the efficient detection of the reactionproducts. The low thermal mass of the system ensures rapid heating andcooling rates since there is little material to heat and cool andbecause there is a high surface to volume ratio for thermal transfer.Those skilled in the art have only recently appreciated that rapidheating and cooling improves efficiency and decreases the amount ofextraneous undesirable reactions, and that certain reactions could beperformed with high thermal exchange rates.

The objectives of the invention are to greatly increase thermal exchangerates in chemical processes, such as PCR (up to 10× faster heating andcooling rates), to optimize temperature uniformity of the reactants, toaccommodate high thermal expansion coefficients to minimizethermal-induced mechanical stress; maximize optical excitationefficiency (long optical pathlength), to maximize optical detectionsensitivity (maximize photon collection volume), to maximize faultdetection capability, to minimize computer overhead of the hostinstrument, and to minimize overall power consumption of the instrumentvia independent, modular, intelligent reaction units supported by apowerful instrument platform and technology for long term versatility.

The objectives of the invention are attained via self-containedheat-exchanging units with optics. Each modular, heat-exchanging unitconstitutes a single reaction site. Overall, each unit comprises (a) athermal sleeve for receiving a reaction vessel, wherein the thermalsleeve has integral heating element(s) for heating a reaction mixturecontained in the vessel; (b) a pair of optics assemblies thatincorporate solid-state excitation and detection sources; (c) a coolingsystem, e.g., a fan or Peltier device, for cooling the reaction mixture;and (d) a housing for the thermal sleeve, optics assemblies, and coolingsystem.

The modular, heat exchanging unit(s) may be supported by a base supporthaving one or multiple circuit boards with microcontrollers formonitoring control of the light excitation and detection sources and forcommunicating with a host computer. The base support may include a maincontroller board which communicates with the optics assemblies, thermalsleeve, and reaction chamber to control procedures such as temperatureand optical control; self-diagnostic protocols; and receipt, storage andprocessing of data, wherein each heat exchanging unit may be separatelycontrolled or the cluster of units may be under a single set ofcontrols.

Reaction Vessel

FIG. 1 shows a partially exploded view of a reaction vessel according tothe present invention. The vessel includes a reaction chamber forholding a sample for chemical reaction. The vessel is designed foroptimal thermal conductance and for efficient optical viewing of thereaction product. The thin shape of the vessel contributes to optimalthermal kinetics by providing large surfaces for thermal conduction andfor contacting temperature-regulating elements, e.g., thermal plates. Inaddition, the minor or major walls provide windows into the chamber sothat the entire reaction mix can be optically interrogated. In addition,the vessel is suitable for a wide range of reaction volumes.

In more detail to the components shown in FIG. 1, a reaction vessel (2)has a housing 6 defining a port (4) and a channel (8) connecting theport (4) to a reaction chamber (10). A seal cap (12) for sealing theport (4) is attached to the housing (6) by a flexible arm (14). The cap(12) is insertable into the port (4) to engage channel (8). A rigidsupport frame (16) and thin flexible walls (18), shown in FIG. 1exploded from the frame, define the chamber (10), wherein the flexiblewalls (18) are coupled to opposite sides of the frame (16). On the rigidframe (16) are reflective faces (20) which bounce back light transmittedfrom the chamber (10), allowing for increased detection of signal.

In using the reaction vessel (2), a sample added to port (4) flowsthrough the channel (8) and into chamber (10). In the chamber (10) thesample is introduced to chemicals for reacting. The major walls (18) ofthe chamber (10) are made to press against heating or cooling elements,e.g., thermal plates, and the walls (18) conform to the element surface.The sample is exposed to variations in temperature by activating theheating/cooling element. The reaction and/or reaction products areoptically viewed.

The thin, flexible walls (18) which define the sides of the chamber (10)facilitate optimal thermal conductance to the chemicals contained in thechamber (10). The flexible nature of the walls (18) allow for maximumcontact with a heating or cooling source. The walls are conformable tothe surface of an external thermal element such that the surface of theflexible walls may adapt to the shape of the external heating/coolingelement surface in a manner that avoids or minimizes gaps between thetwo surfaces. Furthermore, the flexible wall continues to conform to thethermal surface if the surface shape changes during the course of theheat exchanging operation. For example, as the heating element expandsdue to the increased temperature, the chamber wall also expands tomaintain optimal contact with the heating element. Also, if the wallsexpand due to an increase of internal pressure within the chamber, thewalls do not become rigid, but remain conformed to the heating orcooling surface. Contact may be made by biasing the thermal sourceagainst the walls and/or by pressing the walls against the thermalsurface.

FIGS. 2a and 2 b demonstrate the contact a reaction vessel (46) makeswith a thermal sleeve (32) to form a heat exchanging module (30). InFIG. 2a, the thermal sleeve (32) includes thermal plates (36), (38)which are in a relaxed position with opening (34) between the plates.However, as depicted in FIG. 2b, when the reaction vessel (46) withflexible walls (48) is inserted in the opening between the plates (36),(38), the plate surfaces (40), (42) fully engage the chamber walls (48).In this activated position, minimal or no gaps are found between theplate surfaces and the chamber walls (48). The plates (36), (38) aremade to bias against the walls (48) by springs (44). In the alternative,the chamber walls (48) are made to press against the thermal plates(36), (38). The conformable chamber walls (48) mold to the shape of theheating surfaces to provide maximum thermal contact between surfaces.

Further to the ability of the reaction vessel to optimize thermalkinetics, the flexible walls (48) are of low thermal mass to permitrapid heat transfer. FIG. 3 shows a top view of a reaction vessel (50)which is in intimate contact with heating elements (52) and surroundedby cooling chamber (54). The thickness of each flexible wall ispreferably between about 0.0001 to 0.020 inch, more preferably 0.0005 to0.005 inch, and most preferably 0.001 to 0.003 inch. In order to achievethis thinness, the wall may be a film, sheet, or a molded, machinedextruded or cast piece, or other convenient thin and flexible structure.

Referring again to FIG. 1, the material composing the flexible walls(18) may be a polyalcohol including polypropylene, polyethylene,polyester, and other polymers, laminates or homogenous polymers, metalsor metal laminates, or other materials which may be thin, flexible,conformable and permit high heat transfer and is preferably in the formof a film or sheet. Where the rigid frame (16) of the vessel whichsupports the walls (18) is a particular material, such as polypropylene,the walls (18) are preferably the same material, such as polypropylene,so that the heat expansion and cooling rates of the walls (18) are thesame as the frame (16). Therefore, undue heat or cooling-inducedstresses on the materials are minimized so that wrinkling of the walls(18) is avoided during multiple temperature cycling.

Although the flexible walls are preferred in some embodiments, the wallswhich contact the heating elements may also be rigid and flat tocommunicate with a rigid and flat heater. Whether rigid or flexible, thewalls which contact the heating elements are typically the major wallsof the chamber. The chamber also has a plurality of minor walls providedby the rigid frame which support the major walls.

FIGS. 4a, b, c, d show another embodiment of a reaction vessel (60) withminor walls of the reaction chamber (76) angled to optimize opticalviewing. As shown in FIGS. 4a and 4 b, five contiguous minor faces orwalls (62), (64), (66), (68), (70) couple together two opposing majorfaces or walls (72), (74) to form the reaction chamber. Minor walls(64), (66) are coupled together at an angle. As shown in FIG. 4c, theangled walls (64), (66) may define the bottom portion of the reactionchamber (76), and back walls (78) may define the top portion of thechamber. A channel (80) leads to the chamber (76). The channel andchamber including the backwalls may optionally be a separate pieceinserted into the main body of the reaction vessel. The channel (80)leading to the chamber (76) may serve a variety of functions includingproviding a conduit for filling the chamber, such as by bottom filling,or providing an area to hold overfilled reagents and purged air.

The angled walls (64), (66) may be joined to form a “V” shaped point(82), especially on the bottom of the chamber (76) to allow for easierfilling by reducing or eliminating bubble formation. Alternatively, theinterface of the angled walls need not connect to form a point, but maybe separated by an intermediary portion, such as another minor wall orvarious mechanical or fluidic features which do not significantlyinterfere with the thermal and optical performance of the chamber (76).For example, the angled walls may meet at a port which leads to anotherprocessing area in communication with the reaction chamber (76), such asan integrated capillary electrophoresis area.

The reaction vessel also includes a port for adding liquids and removingair from the chamber (76). The port allows access of a pipette tipthrough the channel (80) into the interior of the chamber (76), forexample, to the bottom of the chamber to enable bottom-up filling. Theport may also permit other conventional methods of sample introduction,such as through an automated fluid injection system or through a fluidicmanifold which optionally is an integral part of the reaction vessel.The vessel may also be one aspect of a larger device which processes thefluid prior to the fluid flowing through the port and into the chamber.One example of a larger device is a disposable fluidic cartridge asdisclosed in copending U.S. patent application Ser. No. 08/998,188 filedDec. 24, 1997, the disclosure of which is incorporated by referenceherein.

The external terminus of the port is designed to be sealed, preferablyby accepting a seal cap (84), as shown in FIG. 4d. The cap (84) providesa means for sealing the port after filling to provide a barrier betweenthe thermally controlled interior reaction volume and the non-thermallycontrolled environment to inhibit contamination of the sample, toprevent evaporation of fluid during heating of the sample, and the like.In various embodiments anticipated by the present invention, the sealmay be a snap-on cap, a screwtop, or other specialized closure as neededfor the selected analytical protocol. Such a cap may be composed of anyconvenient material such as polypropylene or an elastomer, such asSantoprene™ (trademark of Monsanto Corporation, located in San Louis.Mo.). In one embodiment, the chamber may be further sealed from theexterior environment by the heating of plastic material on or composingthe top minor wall. In another embodiment the seal is created by a dropof oil placed on top of an aqueous sample to prevent evaporation of thesample.

Referring again to FIG. 1, the cap (12) may also be a plug which isinserted into the channel (8) in communication with the reaction chamber(10) such that the plug creates an increase in pressure within thechamber (10). The resulting increased pressure causes outward expansionof the flexible walls (18) to force the walls against the externalheating units creating a better contact between the walls and theheating elements. The increased pressure may also allow for a solutionin the chamber to remain in a liquid state without going into a gaseousstate at certain high temperatures. This property is in accordance withthe theoretical principle, PV=nRT, where P is pressure, V is volume, nis moles, R is a constant and T is temperature.

The reaction vessel may be configured to optimize the visualization ofthe reaction in the chamber. To this end one, two or more minor walls ofthe chamber comprise the optical windows. Where two windows are present,one window may serve as a light excitation entry port and the secondwindow for detection of light emitted from the reaction chamber. Inanother embodiment, both windows serve for excitation and detection fortwo light paths. In the first light path, light is radiated through thefirst window and detected through the second window. In the second lightpath, light is emitted through second window and detected through thefirst window. The window faces may be offset at an angle selected tomaximize the detection process.

FIG. 5 shows a reaction vessel (90) associated with an external opticalsystem (92). Optical system (92) is designed to illuminate the lighttransmissive, minor wall (94) with optical excitation radiation fromindividual light source (98) and to detect light emitted from thechamber through the light transmissive, minor wall (96) with detector(104). In the alternative, both adjacent optical walls (94), (96) mayreceive radiation from respective light sources (98), (100) andobservation by detectors (102), (104), where the excitation light whichis radiated through each wall is a different wavelength and lightdetected at each wall is also a different wavelength. Exemplary paths ofdiffering wavelengths of excitation and detection light are shown byarrows in FIG. 5. Each of the walls (94), (96) may additionally havelenses (106) directly molded into its surface to direct the light.Optimum optical sensitivity is attained by maximizing the opticalsampling path-length of both the light beams exciting the chemicalmolecules and the emitted light that is detected to generate the opticalsignal.

Where excitation and detection occurs at different walls as in FIG. 5,it is usually preferred that the optical walls (94), (96) are offset atan angle (A). The preferred angle is about 90°. A 90° angle betweenexcitation and detection optical paths assures that a minimum amount ofexcitation radiation entering through one optical wall will exit throughthe other optical wall. Also the 90° angle permits a maximum amount ofemitted radiation, e.g. fluorescence, to be collected through thedetection window. In other embodiments, the angle between adjacentoptical walls is larger or smaller than 90°, depending, inter alia, onthe efficiency and sensitivity of the excitation and detection optics.For example, where a detection system effectively discriminates betweenexcitation and emitted light, an angle of less than 90° between wallsmay be desired. Conversely, where a detection system fails toefficiently discriminate between excitation and emitted light, an anglegreater than 90° may be of interest.

One or more light transmissive elements may be present on the opticalwalls. The optical elements may be designed, for example, to maximizethe total volume of solution which is illuminated by an LED excitationsource, to focus an optical excitation source on a specific region ofthe reaction chamber, or to collect as much fluorescence signal from aslarge a fraction of the reaction chamber volume as possible. Inaddition, gratings for selecting specific wavelengths, filters forallowing only certain wavelengths to pass, and multiple lenses orfilters optimized for multiple excitation sources or detectors may beused. In another embodiment, the opposite wall may be optimized tocollect and focus the maximum percentage of emitted fluorescence signalfrom the solution to an array of photodetectors. Alternatively, theoptical walls may be simple, clear, flat windows serving as opticallytransmissive windows. Other elements include colored lenses to providefiltering functions, retro-reflective surfaces, optical gratingsurfaces, etc.

Further to the reaction vessel, the major or minor walls defining thereaction chamber may be adapted for additional optical interrogation.The wall surfaces may be coated or comprise materials such as liquidcrystal for augmenting the absorption of certain wavelengths. Thesurfaces may be used to determine the temperature of the enclosedchemicals by detecting particular absorption bands which reflecttemperature conditions.

Thin films of metals, polymers, and combinations of materials such as inlaminates, not only can be employed in a reaction chamber for thestructural and thermal properties, but for optical properties as well.Thin films constitute materials having a thickness ranging from a fewangstroms to hundreds of microns, and are usually formed with aparticular series of processes familiar to those in the art of vapordeposition, plasma deposition, magnetron and RF sputtering, laserablation, etc. For example, vapor-deposited thin films of silver canaugment the detection and collection of raman (inelastic scattering ofan optically excited source) spectra. This and other materials can bedeposited on a variety of substrates (glass, plastic, silicon, metals,etc.) to be translucent (transmitting) in certain wavelengths at anglesof incidence, and reflective in others. This is the basis of a lot ofoptical materials developments and devices such as dichroic beamsplitters, dielectric band pass filters, neutral density filters, etc.

The use of these capabilities to manufacture films that can be attachedto, used to hermetically seal reaction vessels, or are depositeddirectly onto the wall of a reaction vessel that will be opticallyinterrogated, can result in reaction vessels with specific opticalemission and excitation properties. These thin film processes when usedeconomically, can thereby be used to manufacture reaction vesselsinexpensively resulting in disposable vessels with fine-tuned opticalproperties.

The reaction vessel may be fabricated in various ways. It may be moldedin several pieces which are bonded together or injection molded in onepiece. There are several advantages to the multi-piece design andmanufacturing approach. One benefit is that very thin walls can beachieved where the walls can be consistently produced to be the samesize and shape. Another benefit is that the optical features of thedevice are separated from the fluidic features so that both componentscan be independently designed and optimized. For exampleretro-reflective walls may be made on one or many sides of the chamberto reflect light. A third advantage is that the primary opticalcomponent can be fabricated from a different material than the primaryfluidic component. An additional benefit is that the major surfaces maybe fabricated from a different material than some or all of the minorsurfaces. For example, materials with optimal thermal characteristicsmay be different from those with optimal optical characteristics. Inparticular, the angled optical windows, with or without lightcomponents, could be molded from polycarbonate, which has good opticaltransparency, while the rest of the chamber could be molded frompolypropylene, which is inexpensive and is known to be compatible withthe sensitive PCR reaction. Both pieces can be bonded together in asecondary step. The optical window is press-fitted or bonded into an endof the chamber, preferably the bottom of the chamber.

In one method of fabricating a reaction vessel, the rigid frame ismolded to form a chamber having open sides. The frame is made bystandard injection molding processes for thermal materials. After theframe is made, the major walls are produced by placing and preferablystretching material (e.g., thin plastic films or sheets) over thechamber area. The walls are then bonded to the frame. Where the wallsare a film or sheet, the material may be attached by heat sealing,adhesive bonding, ultrasonic bonding, etc., to the frame.

A chamber in which the major and minor walls are fabricated from thesame material requires that the total surface area of the major surfacesbe at least about twice that of the total surface area of the minorsurfaces where a thermal conductance ratio of 2:1 is desired. On theother hand, if the walls are made of different materials, it is possibleto modify the geometry from that shown since major walls comprised ofmaterials with high thermal conduction could be combined with minorwalls of low thermal conduction. The walls may be fabricated from glassor polymers including polyacrylics, polyamides, polycarbonates,polyesters, and vinyl polymers, or any combination thereof.

An insert separate from the main frame of the reaction vessel may beplaced inside of the vessel to define some of the chamber or otherinternal features. The insert may fill the top of the chamber andprovide some of the walls. The insert may be bonded or preferablypress-fitted into the vessel. The insert may also provide the channel,port, and cap attachment means.

The shape of the chamber may differ according to the particular reactionbeing performed and the associated thermal transfer device. Furthermore,the relationship of the frame to the flexible walls may vary as long asthe frame is coupled to the walls and the walls are accessible tocontact an external thermal source. The reaction vessel may be sized, inparticular in the chamber, to contain volumes from nanoliters tomilliliters, depending upon the desired use. The volume of the chamberis preferably in the range of 1 to 1000 microliters, more preferably inthe range of 1 to 500 microliters, and most preferably in the range of10 to 100 microliters.

In summary of the reaction vessel, the various embodiments have thefollowing characteristics: high surface-to-volume ratio for efficientheating/cooling; thin, low-mass walls; conformable sidewalls to maximizeassociation with heating and cooling system; moldable and bondablematerial where multiple pieces are required; features which accommodatehigh thermal expansion coefficients to minimize temperature-inducedmechanical stress during heat exchanging operations; chemically inertmaterials so that there is no adsorption or reaction with reactants,intermediates, or products, no or minimal inactivation of enzymes bysurface active means, and compatible with glycerol; windows with highoptical clarity for efficient interrogation; long excitation opticalpath lengths; maximized offset between excitation and emission detectionwindows; no or minimal light coupling between excitation and detectiondevices; optical elements such as retro-reflective surfaces; major wallsprecisely mated with module heating/cooling surfaces; a port forintroducing sample and reagents; means to preclude or minimize refluxingduring cycling; efficient removal of air during filling and capping; andsealing of the reaction mixture from external environment.

Thermal Sleeve

The reaction vessel is compatible with a thermal sleeve for heating orcooling the mixture in the chamber. The thermal sleeve is designed toinduce a temperature change in the chamber by making intimate contactwith the walls of the chamber.

FIG. 6 shows a partially exploded view of one thermal sleeve (200) withone heating or cooling plate (202) attached to a support bridge (206)and another plate (204) removed from the support (206). Each of theplates (202), (204) has one face being a contact surface (208) andanother face being a biasing surface (210) with one end (212) of eachplate being slanted towards each other. Each biasing surface (210) maybe biased by a spring (214) with an integral attachment arm (216) forcoupling to support bridge (206) at securing region (218). The plate ispartially inserted through bridge slots (220) and the plate attachmentarm (216) is fastened to the bridge securing region (218). The biasingsurfaces (210) of the plates also have a plurality of electricalconnections (220) which may communicate with heating, cooling orelectrical sources (not shown) or any combination thereof. Whenassembled, the plates are held in opposition by the support bridge toform an opening between the plates for enclosing a reaction chamber.

FIG. 7 demonstrates how a reaction vessel (252) may be enclosed withinthe thermal sleeve to form a heat exchanging module (250). The contactsurface (208) of each plate (202), (204) is made to press against thereaction chamber surfaces in a manner that maximizes thermal contact. Ingeneral, the sleeve may include one or more separate spring-loadedheater plates configured to mechanically bias against the surface, e.g.chamber area, of the reaction vessel. Such a spring-loaded configurationwould simplify mechanical tolerances between the various mechanicalcomponents, i.e. thermal sleeve, vessel, and optical unit.Alternatively, the plates may be made to bias against a reaction chambersurface by mechanical force initiated by other parts or a mechanicalmotor, thermal expansion, pneumatic pressure, such as air flow pressure,hydraulic pressure, and the like. The interior of the heat exchangingsleeve may also be tapered to allow for a snug fit with an insertedreaction vessel. Furthermore, the walls of the inserted chamber areexpanded to also press against the heater plate surfaces.

The shape of the sleeve is designed for optimal thermal contact with areaction chamber. In one embodiment, the opening in the sleeve forcontacting the chamber walls is elongated in the x-direction. Thus, theopening is longer in the direction perpendicular to the length of thechamber. Preferably the shape of the opening is rectangular in crosssection. The ratio of length to width of the surfaces defining theopening may be at least 2:1. Such elongation provides greater contactwith the chamber walls than prior designs where the opening forinserting a vessel is expanded in the z-direction and the opening istypically round or octangular in shape to hold round tubes.

As described in this application, the achievement of rapid heat exchange(heating and/or cooling) from a sample in a reaction tube or vesselrequires a low thermal-mass thermal sleeve assembly together with athin, wide reaction vessel. The fastest thermal cycling instruments todate solve this problem by making the reaction vessel a thin longcylinder, 1 mm in diameter or less. Other fast thermal cyclinginstruments depend on very small liquid sample volumes, which arerelatively easy to heat and cool rapidly. However, these approaches areonly suitable for very small sample volumes.

In contrast, the reaction vessel described here is thin and wide.Instead of increasing the volume capacity by simply making the reactionvessel longer, this application teaches that large heating and coolingrates can also be achieved by properly designed reaction vessels whichare, instead, thin and wide. The counterpart and complementary design ofthe low-mass thermal sleeve assures that the entire assembly, includingthe relatively large sample volumes (up to and over 100 μL), can becontrollably heated and cooled at the maximum rates.

The thermal plates may be made of various materials. In order to ensurethat the inside of the heat exchanging sleeve is resistant to bleach andother cleaning solutions, the interior may be coated or lined with achemically inert material, such as a polytetrafluoroethylene, or theentire sleeve may be fabricated from a chemically stable material, suchas a ceramic or metals such as aluminum nitride, aluminum oxide,beryllium oxide, and silicon nitride. Other materials which may beutilized include, e.g., gallium arsenide, silicon, silicon nitride,silicon dioxide, quartz, glass, diamond, polyacrylics, polyamides,polycarbonates, polyesters, polyimides, vinyl polymers, and halogenatedvinyl polymers, such as polytetrafluoroethylenes. Other possiblematerials include thermocouple materials such as chrome/aluminum,superalloys, zircaloy, aluminum, steel, gold, silver, copper, tungsten,molybdenum, tantalum, brass, sapphire, or any of the numerous ceramics,metals, and synthetic polymeric materials available in the art.

Ceramic plates are preferred because the inside surfaces may beconveniently machined to very high smoothness for high wear resistance,high chemical resistance, and good thermal contact to reaction vessels.Ceramic plates can also be made very thin (between 0.025-0.050 inches)for low thermal mass. A heat exchanging plate made from aluminum orcopper also has high thermal conduction, but a larger thermal mass.

The heating source, such as heating resistors, may be directly screenprinted onto a plate, particularly plates comprising ceramic insulatingmaterials, such as aluminum nitride and aluminum oxide. Screen printingprovides high reliability and low cross-section for efficient transferof heat into the chamber itself. The heating element may also be bakedinside of the ceramic plate. Also, thick or thin film resistors ofvarying geometric patterns may be disposed on the plate walls to providemore uniform heating, for example by having thicker resistors at theextremities and thinner resistors in the middle. Heating elements mayconsist of carbide, tungsten, silver, or other materials which heat whena voltage is applied to the material. One way of heating a metal sleeveis by using a laminated heater source such as an etched-foil heatingelement (Minco Products, located in Minneapolis, MN) attached to thesurface of the heating plates. Optionally, cooling fins, of the same ordifferent material as the body of the chamber, may be brazed, soldered,or epoxied directly over the screen-printed resistors.

The function of the support bridge (206), shown in the embodiment inFIG. 6 and described above, is to serve as a support for one or morethermal sleeve heating or cooling elements and to provide a guide forinserting the reaction chamber into the thermal sleeve. The support mayinclude a slot for inserting the chamber between the thermal plates. Theslot may incorporate mechanical features or a sealing surface whichallows a tight mechanical seal. A consideration in the choice ofmaterial for the support is that its thermal expansion coefficient (TCE)match that of the thermal plates as closely as possible. The materialsof construction recited above for the plate are also useful for thesupport. Appropriate combinations will be apparent to the skilledartisan.

The mechanical transition between the thermal sleeve plate and topsupport is a critical joint. The heating or cooling plate may be cycledmany times (up to 400,000 over a 5 year life), e.g., in PCR applicationsbetween about room temperature, 60° C., and 95° C., while the topsupport may be maintained at a relatively constant temperature. Thermalgradients and stresses are high in this region. Flexible,chemical-resistant adhesives and gasket materials may be used to ensurebonding. Preferred adhesives are epoxy, but a more robust metal sealingtechnique can be used if the thermal plate is metal or ceramic. Anothercriteria for the transition region is that the sealing or bondingmaterial and the method for joining the top support to the thermal plateshould be resistant to bleaches and other cleaning solutions. It isexpected that up to 1000 exposures to cleaning solutions such as 10%bleach and 1% Tween 20 may occur.

The thermal sleeve of this invention has high thermal conduction and lowthermal mass to permit rapid heating and cooling. Further, the thermalsleeve is sufficiently durable for repetitive use (as many as 10,000reaction chamber insertions). The heating elements are integrated intothe sleeve to assure rapid, efficient heating. To maximize coolingefficiency, cooling elements may also be attached to the surface such ascooling fins or thermally conductive elements connected to a secondarycooling source. For example, the sleeve may be thermally connected to aPeltier element or to a heat pipe.

FIGS. 8a, b, c, d illustrate exemplary variations of heating and coolingconfigurations of a thermal sleeve. FIG. 8a is a top view which looksdirectly down into the mouth (262) of the sleeve (260). The sleeve isprovided with cooling fins (264) and integrated heaters (266). In thisembodiment, the sleeve is provided with a thin interior liner (268).FIG. 8b is a front view of the cooling fins (264) shown in FIG. 8a. FIG.8c is a front view of another sleeve (270) with heating element (276)and cooling fins (274). A proportional-to-absolute zero (PTAT)temperature sensor is shown at (272). FIG. 8d is a side view of thesleeve (270) showing screen printed or laminated heating elements (276)beneath the cooling fins (274).

The temperatures of an inserted reaction chamber and/or thermal platesmay be monitored by one or more sensors located on the thermal sleeve.In order to achieve the desired 0.5-1.0 ° C. temperature accuracy,silicon-based, proportional-to-absolute zero (PTAT) thermal sensors maybe used. The output of the sensors is linearly proportional totemperature. High precision PTAT temperature sensors can be very small,e.g. 0.5×0.5×1.0 mm. Alternatively, thermistors, thermocouples andresistance temperature detectors (RTD), especially RTD's made fromplatinum, copper, nickel and nickel-iron may be used. These sensors areeasily affixed to the trailing edge of the heat exchanging reactionchamber.

The thermal sleeve is also adapted for optical interrogation of thecontents in situ and may incorporate various features, such as lensesand filters, to facilitate optical visualization. In one embodiment, atleast two surfaces of the sleeve are optically transmissive, preferablyforming the bottom of the sleeve adjacent to the optical windows of aninserted reaction vessel. An important criteria for the window materialis transparency or translucency of the window. The window may alsosimply be an opening in the sleeve through which the reaction chambermay be viewed. In one embodiment, the sleeve is open at the bottom sothat a portion of an inserted chamber may extend below the sleeve fordirect optical interrogation. Where the window is a particular material,it is preferred that the window be as close a match as possible betweenthe coefficients of thermal expansion (TCE) of the sleeve and thewindow. For example, a glass having a TCE closely matches that of aceramic sleeve may be chosen. A plastic window is more suitable if thethermal sleeve is metal. The window material should also be stable tobleach and other cleaning solutions.

The mechanical transition between window and thermal elements is acritical joint. It is desirable to maintain the optics package at arelatively low temperature, or at least constant temperature, while thethermal sleeve is temperature cycled many times. Thermal gradients andstresses are high in this region. Another criteria for the transitionregion is that whatever sealing or bonding material and method forjoining the optical window to the thermal sleeve is used should beresistant to bleaches and other cleaning solutions. In particular, it isenvisioned that the inside of the thermal sleeve will be periodicallycleaned by pipetting in dilute bleach solutions, followed by water,followed by an alcohol dry. These solutions will be in direct contactwith the joint between the optical window and the thermal sleeve. Thistransition also significantly effects illumination and light-gatheringfunctions of the device.

Controlled Heat Exchanging Unit

The optics assembly may be fabricated in a unit which is configured toaccept a thermal sleeve. The unit may additionally have systems formaintaining the environmental temperatures, such as a cooling system,and various control mechanisms to regulate the operations beingperformed within the sleeve.

In FIG. 9, a heat exchanging unit (600) is shown with a housing (602)and associated operational elements. A processing area (604) is adaptedto accept a thermal sleeve (630) and reaction vessel (632) describedsupra. The processing area (604) is in pneumatic communication with acooling fan (606) by an inlet channel (608) and with an outlet channel(610) leading from the processing area (604) to an outlet port (612).When the vessel (632) is inserted into the sleeve (630), the reactionchamber is cooled by the cooling air circulating from the fan (606) toinlet channel (608), to processing area (604). Thereafter, the airtravels through outlet channel (610) to exit the housing at port (612).In addition, the inserted reaction chamber is in optical communicationwith an optics assembly (620) that includes optics emission anddetection blocks coupled to circuit boards (622) for controlling theoptics.

The optical assembly (620) includes lensing elements, such as lightcollimating, e.g. light-pipe, and focussing elements, with transmissiveand reflective filters, gratings, photodiodes, fresnel lenses and thelike, as needed, which may be attached to a circuit board which maycontain LEDs and photodetectors. The lensing components may be injectionmolded from a transparent plastic such as polycarbonate or othermoldable, optically clear plastic or glass. The lensing elements connectthe reaction chamber windows to the excitation and detection opticalcomponents. The lensing elements incorporate and interface with filtersand with the optical excitation and detection circuit boards (622) whichcontain the solid-state LEDs and photodetectors.

Solid-state LED's and photodetectors are optionally assembled onto asmall circuit board located below the lensing components. This is asimple board, fitted with alignment features to accurately position theexcitation sources and detectors with respect to the lensing elementsand the reaction chamber. An edge-connector or flex-connector provideselectrical connection between the optical board and the adjacentcontroller board.

The housing (602) may be machined from aluminum and anodized, moldedfrom a rigid, high-performance plastic, or other conventional techniquesand materials. The primary functions of the housing are to provide aframe for holding the thermal sleeve, top support, and optics assembliesand to provide flow channels and ports for directing cooling fluid, e.g.air, and efficiently guiding the fluid flow across the surface of thethermal sleeve/reaction chamber.

The heat exchanging unit preferably includes a cooling source, such asgas diffusing plates or other air flow distributing structures, forassuring uniform air flow around the thermal sleeve, a fan for blowingcool air over the sleeve, a Peltier device, liquid cooling such as wateror a compressed gas cooling source, or the like. The cooling element maybe directly formed in the housing or be fabricated independently fromthe housing and assembled into the housing at a later time. For example,each thermal sleeve in an array of heat exchanging assemblies maycommunicate with a cooling element. In addition, ports located in thehousing may provide coolant air inlet and outlet ports. The ports mayalso be sealed to a base-plate using suitable sealants, where the portsinterface with inlet and outlet ports in the base plate.

FIGS. 10a and 10 b show an alternative embodiment of a heat exchangingunit (650). The unit (650) has thermal sleeve (670) (shown partiallyexploded from the unit) with thermal plates attached to a top support(674) for mating with the housing (652). Housing (652) has air inlet andoutlet port (654), a support (656) with air diffusing plates (658) andan optics module (660) with attached circuit board (662). The directionof cooling air flow is shown by the arrows.

The entire electronic control of each heat exchanging unit may beincorporated into one or two circuit boards or chips attached to thesides of the housing. In FIG. 10b, the thermal sleeve (670) with topsupport (674) is shown partially exploded out of the housing (652).Optics module (660) and optics circuit board (662) interfaces with apair of controller boards (644). The circuit board (662) and controllerboards (644) may be fabricated using high reliability, low-profilesurface-mount technology. The controller boards (644) communicate withthe optical board through a slot in the housing and a 90° electricalconnector where the bottom end of the circuit board plugs into anelectrical socket on the base-plate for electrical communication to thecontroller board.

Moreover, multiple heat exchanging units may be grouped together, as inconventional reaction apparatus, such as PCR, for exposing multiplesamples to the same temperature profile, in which case only one unitneed be equipped with electrical control circuitry. However, when it isdesired to react multiple samples independently, then independentcontrol of each unit or grouping of units is needed.

In FIGS. 11a and 11 b, a cluster of modular, heat-exchanging units arearranged in an array. FIG. 11a shows one embodiment of a heat exchangingsystem (700) with four modular units (710), one of which is shown with aside panel removed to expose the internal unit, on a base support (702).FIG. 11c shows another embodiment of a heat exchanging system (800) witheight modular units (810) arranged on a base support (802). In FIG. 11aand in the cross-sectional view shown in FIG. 11b, each unit (710) hasoptics circuit boards (712) which interface with the “mother” commandunit (704), e.g., a common controller board. The single controller boardhas several, e.g. four, circuit boards (not shown) so that the “mother”processor board (704) controls the cluster of units (710). A systempower converter (706) supplies power to the units (710).

Alternatively, as shown in FIG. 11c, the optics circuit board (812) ofeach unit (810) interfaces with an individual controller board (820) sothat each unit has its own controller board. In FIG. 11c a thermalsleeve (814) with top support (816), optics assembly (818) and opticalcircuit board (812) are shown removed from the base (802). In an arrayformat, the gap between the heat exchanging units may be sealed by agasketed plate covering the entire array. The top support and thegasketed plate may be configured to form a flush surface for amulti-unit array. The gasket material is preferably resistant to bleachand other cleaning solutions.

Referring again to FIG. 11a, the base support (702) for the array ofmodular units (710) may provide several functions. For example, the basemay allow for physical mounting of the units, housing of a controllerboard (704), and electrical connection between the units and a hostcomputer. A multi-function electrical connector may also serve as thephysical mount.

The footprint of the controlled heat exchanging unit is designed to beeasily assembled into 2-dimensional arrays. In addition, the closespacing in one direction allows the use of linked linear arrays ofunits, if desired. In one embodiment, the overall dimensions of eachmodular unit are approximately 9×40×50 mm. The narrow dimension is smallenough to allow, for example, 8 units to be grouped together, ifdesired, in a reasonable (72 mm) length, suitable for interfacing withstandard commercially available multi-pipettes which have 9 mmcenter-to-center spacing for convenient loading of sample and chemicals,if needed.

The thermal reaction apparatus may find use in many applications. Theapparatus of the invention may be utilized to perform chemical reactionson a sample, for example, polymerase chain reaction (PCR). Each unit isprovided, either directly or within a separate, insertable reactionvessel, with reagents required for the reaction. For example, inperforming a polymerase chain reaction, the chamber of the vessel mayinclude a sample polynucleotide, a polymerase such as Taq polymerase,nucleoside triphosphates, a first primer hybridizable with the samplepolynucleotide, and a second primer hybridizable with a sequencecomplementary to the polynucleotide. Some or all of the requiredreagents may be present in the reaction chamber as shipped, or they maybe added to the sample and then delivered through the inlet port to thechamber, or the reagents may be delivered to the chamber independentlyof the sample. The polymerase chain reaction may be performed accordingto methods well known in the art.

Although polynucleotide amplification by polymerase chain reaction hasbeen described herein, it will be appreciated by persons skilled in theart that the devices and methods of the present invention may beutilized equally effectively for a variety of other polynucleotideamplification reactions and ligand-binding assays. Such additionalreactions may be thermally cycled, such as the polymerase chainreaction, or they may be carried out at a single temperature, e.g.,nucleic acid sequenced-based amplification (NASBA). Moreover, suchreactions may employ a wide variety of amplification reagents andenzymes, including DNA ligase, T7 RNA polymerase and/or reversetranscriptase, among others. Additionally, denaturation ofpolynucleotides can be accomplished by known chemical or physicalmethods, alone or combined with thermal change. Polynucleotideamplification reactions that may be practiced in the apparatus of theinvention include, but are not limited to: (1) target polynucleotideamplification methods such as self-sustained sequence replication (3SR)and strand-displacement amplification (SDA): (2) methods based onamplification of a signal attached to the target polynucleotide, such as“branched chain” DNA amplification; (3) methods based on amplificationof probe DNA, such as ligase chain reaction (LCR) and QB replicaseamplification (QBR); (4) transcription-based methods, such as ligationactivated transcription (LAT) and nucleic acid sequence-basedamplification (NASBA); and (5) various other amplification methods, suchas repair chain reaction (RCR) and cycling probe reaction (CPR).

In addition to the aforementioned gene or target amplification methods,other chemical or biochemical reaction applications are anticipated. Forexample, temperature controlled lysis of cells is another application ofthe intended invention, which may or may not compliment gene or targetamplification methods described above. In many cases, this isaccomplished by raising the temperature of the solution containing thecell to 37° C. for a few minutes to allow the action of proteolyticenzymes followed by raising the temperature and holding at 95° C. Aftera few seconds to minutes, the cell is lysed and the target component,such as nucleic acid, is released and can then be further processed,e.g., amplified. In other applications, it may be desired to immediatelystop any further chemical reactions immediately after the lysis bylowering the temperature to 0° to 4° C., such as in the case whenstudying the mRNA expression state using rapid thermal polymerase chainreaction. The rapid thermal ramping as provided by this apparatusenables such functionality.

Furthermore, the disclosed apparatus can be utilized to control andinterrogate chemical reactions. In enzyme kinetic studies, for example,it is advantageous to hold the test reaction mixture at a reducedtemperature, such as 0° C.-4° C., before starting the reaction, and thento quickly bring the reaction mixture from this reduced holdtemperature, e.g. 4° C., to an optimal reaction temperature. Unwantedside reactions occurring at intermediate temperatures are reduced oreliminated, allowing for more accurate measurements and higher purity ofproduct. Moreover, this approach can be extended to more complexchemical and biochemical reactions that can be controlled and studied byenabling changes to multiple different temperatures, or to periodicallyreduce the temperature to stop the reactions.

Such temperature control can be exploited for ligand binding reactionssuch as fluorescence homogenous immunoassays. Because the reaction startevent can be so precisely executed and the subsequent reaction holdtemperature accurately controlled without thermal gradients, betterassay performance may be achieved. Other applications of the inventionare intended to be within the scope of the invention where thoseapplications require the transfer of thermal energy to a chemicalreaction.

The present invention has been described above in varied detail byreference to particular embodiments and figures. However, it is to beunderstood that modifications or substitutions may be made to thedevices and methods described based upon this disclosure withoutdeparting from the broad scope of the invention. Therefore, the scope ofthe invention should be determined by the following claims and theirlegal equivalents.

What is claimed is:
 1. An apparatus for heating and opticallyinterrogating a reaction mixture, the apparatus comprising: a) a vesselhaving a chamber for holding the reaction mixture, the chamber beingdefined by two opposing major walls and a plurality of minor wallsjoining the major walls to each other, wherein at least two of the minorwalls are light transmissive to provide optical windows to the chamber;b) at least one heating surface for contacting at least one of the majorwalls; and c) optics for optically interrogating the chamber while theheating surface is in contact with at least one of the major walls, theoptics comprising at least one light source for transmitting light tothe chamber through a first one of the light transmissive walls and atleast one detector for detecting light exiting the chamber through asecond one of the light transmissive walls.
 2. The apparatus of claim 1,wherein the heating surface comprises a surface of a plate, the platehaving at least one heating resistor coupled thereto.
 3. The apparatusof claim 2, wherein the plate comprises a ceramic material, and whereinthe resistor is screen-printed on the plate.
 4. The apparatus of claim1, wherein the at least one major wall contacted by the heating surfacecomprises a sheet or film sufficiently flexible to conform to theheating surface.
 5. The apparatus of claim 1, wherein the apparatusincludes at least two heating surfaces defined by opposing platespositioned to receive the vessel between them such that the platescontact the major walls, the apparatus includes means for heating theplates, and the optics are positioned to interrogate the chamber throughat least one window or opening between the plates.
 6. The apparatus ofclaim 5, further comprising at least one support for holding the platesin an opposing relationship to each other, the support including a slotfor inserting the chamber of the vessel between the plates.
 7. Theapparatus of claim 6, further comprising at least one spring for biasingat least one of the plates against one of the major walls.
 8. Theapparatus of claim 1, wherein: i) the apparatus includes at least twoheating surfaces positioned to receive the vessel between them such thatthe heating surfaces contact the major walls; ii) the vessel includes: arigid frame providing the minor walls; two sheets or films attached toopposite sides of the rigid frame to form the two opposing major walls,the sheets or films being sufficiently flexible to conform to theheating surfaces; a port for introducing fluid into the chamber; and achannel connecting the port to the chamber; and iii) the apparatusfurther comprises a plug that is insertable into the channel to increasepressure in the chamber, whereby the pressure increase forces the majorwalls against the heating surfaces.
 9. The apparatus of claim 1, whereinthe optics include: i) a plurality of light sources and filters fortransmitting different wavelengths of excitation light to the chamber;and ii) a plurality of detectors and filters for detecting differentwavelengths of light emitted from the chamber.
 10. The apparatus ofclaim 1, wherein the at least one heating surface is provided by athermal sleeve for receiving the vessel, the sleeve having at least oneheating element for heating the surface, and the sleeve being open atits bottom so that when the vessel is inserted in the sleeve, a portionof the vessel extends below the sleeve for optical interrogation of thechamber through the at least two light transmissive walls.
 11. Theapparatus of claim 1, wherein the at least one heating surface isprovided by a thermal sleeve for receiving the vessel, the sleeve havingat least one heating element for heating the surface, and the sleevehaving at least one window or opening providing optical access to thelight transmissive walls.
 12. The apparatus of claim 1, wherein theapparatus includes at least two heating surfaces provided by opposingplates positioned to receive the vessel between them such that theplates contact the major walls, each of the plates has a heating elementcoupled thereto, the plates, heating elements, and optics areincorporated into a heat-exchanging unit, the apparatus furthercomprises a base support for receiving the heat-exchanging unit, and thebase support includes at least one controller for controlling theoperation of the heat-exchanging unit.
 13. The apparatus of claim 12,wherein the heat-exchanging unit further comprises: i) a housing forholding the plates, heating elements, and optics; and ii) a coolingelement disposed within the housing for cooling the chamber.
 14. Theapparatus of claim 13, wherein the cooling element comprises a fan forblowing cooling air.
 15. The apparatus of claim 12, wherein the basesupport is constructed to receive and independently control a pluralityof such heat-exchanging units.
 16. The apparatus of claim 15, furthercomprising a computer connected to the base support.
 17. The apparatusof claim 1, wherein the light transmissive walls are angulary offsetfrom each other.
 18. The apparatus of claim 1, wherein the lighttransmissive walls are angularly offset about 90° from each other, andwherein the optics provide about a 90° angle between optical excitationand detection paths.
 19. The apparatus of claim 1, further comprisingoptical elements or coatings on the light transmissive walls forallowing only certain wavelengths of light to pass through the lighttransmissive walls.
 20. The apparatus of claim 1, wherein each of thelight transmissive walls has a lens molded into its surface.
 21. Theapparatus of claim 1, wherein the vessel includes at least four minorwalls defining the chamber, at least two of the minor walls being thelight transmissive walls providing the optical windows to the chamber,and at least two other of the minor walls being retro-reflective wallsfor reflecting light in the chamber.
 22. The apparatus of claim 1,wherein the apparatus includes a heat source for heating the surface.23. The apparatus of claim 22, wherein the heating surface comprises thesurface of a plate, and wherein the heat source comprises a heatingresistor or etched-foil heating element coupled to the plate.
 24. Theapparatus of claim 1, further comprising a cooling element for coolingthe chamber.
 25. The apparatus of claim 24, wherein the cooling elementcomprises a fan for blowing air.
 26. An apparatus for heating andoptically interrogating a reaction mixture contained in a vessel, thevessel having a chamber defined by two opposing major walls and aplurality of minor walls joining the major walls to each other, at leasttwo of the minor walls being light transmissive to provide opticalwindows to the chamber, the apparatus comprising: a) at least oneheating surface for contacting at least one of the major walls; and b)optics positioned to optically interrogate the contents of the chamberwhile the heating surface is in contact with at least one of the majorwalls, the optics comprising at least one light source for transmittinglight to the chamber through a first one of the light transmissive wallsand at least one detector for detecting light exiting the chamberthrough a second one of the light transmissive walls.
 27. The apparatusof claim 16, wherein the heating surface comprises a surface of a plate,the plate having at least one heating resistor coupled thereto.
 28. Theapparatus of claim 16, wherein the apparatus includes at least twoheating surfaces defined by opposing plates positioned to receive thevessel between them such that the plates contact the major walls, theapparatus includes means for heating the plates, and the optics arepositioned to interrogate the chamber through at least one window oropening between the plates.
 29. The apparatus of claim 18, furthercomprising at least one support for holding the plates in an opposingrelationship to each other, the support including a slot for insertingthe chamber of the vessel between the plates.
 30. The apparatus of claim16, wherein the optics include: i) a plurality of light sources andfilters for transmitting different wavelengths of excitation light tothe chamber; and ii) a plurality of detectors and filters for detectingdifferent wavelengths of light emitted from the chamber.
 31. Theapparatus of claim 16, wherein the at least one heating surface isprovided by a thermal sleeve for receiving the vessel, the sleeve havingat least one heating element for heating the surface, and the sleevebeing open at its bottom so that when the vessel is inserted in thesleeve, a portion of the vessel extends below the sleeve for opticalinterrogation of the chamber through the at least two light transmissivewalls.
 32. The apparatus of claim 16, wherein the at least one heatingsurface is provided by a thermal sleeve for receiving the vessel, thesleeve having at least one heating element for heating the surface, andthe sleeve having at least one window or opening providing opticalaccess to the light transmissive walls.
 33. The apparatus of claim 16,wherein the apparatus includes at least two heating surfaces provided byopposing plates positioned to receive the vessel between them such thatthe plates contact the major walls, each of the plates has a heatingelement coupled thereto, the plates, heating elements, and optics areincorporated into a heat-exchanging unit, the apparatus furthercomprises a base support for receiving the heat-exchanging unit, and thebase support includes at least one controller for controlling theoperation of the heat-exchanging unit.
 34. The apparatus of claim 33,wherein the heat-exchanging unit further comprises: i) a housing forholding the plates, heating elements, and optics; and ii) a coolingelement disposed within the housing for cooling the chamber.
 35. Theapparatus of claim 34, wherein the cooling element comprises a fan forblowing cooling air.
 36. The apparatus of claim 33, wherein the basesupport is constructed to receive and independently control a pluralityof such heat-exchanging units.
 37. The apparatus of claim 36, furthercomprising a computer connected to the base support.
 38. The apparatusof claim 26, wherein the light transmissive walls are angularly offsetfrom each other by about a 90° angle, and wherein the optics provideabout a 90° angle between optical excitation and detection paths. 39.The apparatus of claim 26, wherein the apparatus includes a heat sourcefor heating the surface.
 40. The apparatus of claim 39, wherein theheating surface comprises the surface of a plate, and wherein the heatsource comprises a heating resistor or etched-foil heating elementcoupled to the plate.
 41. The apparatus of claim 26, further comprisinga cooling element for cooling the chamber.
 42. The apparatus of claim41, wherein the cooling element comprises a fan for blowing cooling air.43. An apparatus for heating and optically interrogating a reactionmixture contained in a vessel, the vessel having a chamber for holdingthe mixture, the chamber being defined by a plurality of walls, at leasttwo of the walls being light transmissive to provide optical windows tothe chamber, the apparatus comprising: a) at least one heating surfacefor contacting at least one of the plurality of walls; b) at least oneheat source for heating the surface; and c) optics positioned tooptically interrogate the chamber while the heating surface is incontact with at least one of the plurality of walls, the opticscomprising at least one light source for transmitting light to thechamber through a first one of the light transmissive walls and at leastone detector for detecting light exiting the chamber through a secondone of the light transmissive walls.
 44. The apparatus of claim 43,wherein the heating surface comprises a surface of a plate, and whereinthe heat source comprises at least one heating resistor coupled to theplate.
 45. The apparatus of claim 43, wherein the apparatus includes atleast two heating surfaces defined by opposing plates positioned toreceive the vessel between them such that the plates contact opposingwalls of the chamber, the heat source comprises heating resistorscoupled to the plates, and the optics are positioned to interrogate thechamber through at least one window or opening between the plates. 46.The apparatus of claim 45, further comprising at least one support forholding the plates in an opposing relationship to each other, thesupport including a slot for inserting the chamber of the vessel betweenthe plates.
 47. The apparatus of claim 43, wherein the optics include:i) a plurality of light sources and filters for transmitting differentwavelengths of excitation light to the chamber; and ii) a plurality ofdetectors and filters for detecting different wavelengths of lightemitted from the chamber.
 48. The apparatus of claim 43, wherein the atleast one heating surface is provided by a thermal sleeve for receivingthe vessel, the sleeve having at least one window or opening providingoptical access to the light transmissive walls, and the optics beingpositioned to interrogate the chamber through the at least one window oropening in the sleeve.
 49. The apparatus of claim 45, furthercomprising: i) a housing for holding the plates, heat source, andoptics; and ii) a cooling element disposed within the housing forcooling the chamber.
 50. The apparatus of claim 49, wherein the housing,plates, heat source, optics, and cooling element form a modularheat-exchanging unit, and wherein the apparatus further comprises: abase support for receiving a plurality of such modular heat-exchangingunits; and at least one controller for independently controlling theoperation of each heat-exchanging unit.
 51. The apparatus of claim 49,wherein the cooling element comprises a fan for blowing cooling air. 52.The apparatus of claim 43, wherein the light transmissive walls areangularly offset about 90° from each other, and wherein the opticsprovide about a 90° angle between optical excitation and detectionpaths.
 53. An apparatus for heating and optically interrogating areaction mixture contained in a vessel, the vessel having a chamberdefined by two opposing major walls and a plurality of minor wallsjoining the major walls to each other, at least two of the wallsdefining the chamber being light transmissive to provide optical windowsto the chamber, the apparatus comprising: a) at least one plate forcontacting at least one of the major wails; b) at least one heater forheating the plate; and c) optics positioned to optically interrogate thecontents of the chamber while the plate is in contact with at least oneof the major walls, the optics comprising at least one light source fortransmitting light to the chamber through a first one of the lighttransmissive walls and at least one detector for detecting light exitingthe chamber through a second one of the light transmissive walls. 54.The apparatus of claim 53, wherein the apparatus includes at least twoplates positioned in an opposing relationship to each other to receivethe vessel between them such that the plates contact the opposing majorwalls of the chamber, the apparatus includes at least two heaters forheating the plates, a first one of the heaters being coupled to a firstone of the plates and a second one of the heaters being coupled to asecond one of the plates, and the optics are positioned to interrogatethe chamber through at least one window or opening between the plates.55. The apparatus of claim 54, further comprising at least one supportfor holding the plates in an opposing relationship to each other, thesupport including a slot a for inserting the chamber of the vesselbetween the plates.
 56. The apparatus of claim 53, wherein the opticsinclude: i) a plurality of light sources and filters for transmittingdifferent wavelengths of excitation light to the chamber; and ii) aplurality of detectors and filters for detecting different wavelengthsof light emitted from the chamber.
 57. The apparatus of claim 54,further comprising: i) a housing for holding the plates, heaters, andoptics; and ii) a cooling element disposed within the housing forcooling the chamber.
 58. The apparatus of claim 57, wherein the housing,plates, heaters, optics, and cooling element form a modularheat-exchanging unit, and wherein the apparatus further comprises: abase support for receiving a plurality of such modular heat-exchangingunits; and at least one controller for independently controlling theoperation of each heat-exchanging unit.
 59. The apparatus of claim 53,wherein the heater comprises a heating resistor coupled to the plate.60. The apparatus of claim 53, wherein the light transmissive walls areangularly offset about 90° from each other, and wherein the opticsprovide about a 90° angle between optical excitation and detectionpaths.